Many non-enveloped viruses cause devastating human diseases. The mechanisms of entry of these viruses into host cells are poorly understood, although there are likely analogies with well-studied enveloped viruses like flu, HIV and herpesvirus. We have studied non-enveloped dsRNA viruses with a single-layered (cytoplasmic polyhedrosis virus - CPV), a double-layered (aquareovirus), and a triple-layered (bluetongue virus - or BTV -- a member of the Orbivirus genus of the Reoviridae family) capsid. Because its cell biology is well studied, and because it has separate attachment and penetration proteins, BTV in particular serves as a good model system for studying cell entry and transport by such viruses. Thus, the long term goal of this project is to uncover, by cryo electron microscopy (cryoEM), the structural basis of non-enveloped virus infection, particularly the processes of viral attachment and entry, as well as cellular transport of progeny viral particles. Our 7A-resolution cryoEM structure of the pre-penetration BTV virion suggests the presence of a central three-helix bundle and 18 amphipathic helices on the surface of the BTV penetration protein (VP5), similar in some respects to Class I fusion proteins of enveloped viruses. This assignment of amino acids to secondary structures constitutes a set of hypotheses begging to be tested at atomic resolution. Moreover, by use of low pH, we have transformed the virus to its penetration state and visualized the blossoming of long 'barbs' that we hypothesize to be unfurled amphipathic helices. Further, we hypothesize involvement of a disulfide bond within VP5 in the unfurling mechanism. Our cryoEM structure also suggests that the cell attachment protein (VP2) has two binding sites, one for sialic acid and one for an unknown target molecule, perhaps an integrin. In addition, our preliminary data shows that non-structural protein NS1 has a role on virus release and forms highly ordered helical tubules in a Zn2+-dependent manner, thus providing an opportunity for structural studies to explain its role. The proposed studies will test these hypotheses by carrying out four specific aims: (1) By determining the atomic structure of the native BTV virion, we will test our hypothesis that the VP5 penetration protein of the non-enveloped BTV virus has a three-helix bundle at its core, 18 amphipathic helices on its surface, and a disulfide bond in a critical position. (2) From the atomic structure of the native BTV virion in the presence of sialic acid, we will test our hypothesis that the VP2 attachment protein has a sialic-acid binding site. (3) By determining the structure of the (blossomed) penetration state (low pH) of the BTV particle, we will test our hypothesized unfurling mechanism. Moreover, we will carry out structure-based mutagenesis studies to complement structural studies to establish mechanisms for triggering the unfurling. (4) From the atomic structure of BTV non-structural protein NS1 in helical tubules, followed by functional and structure-based mutagenesis experiments, we will learn how NS1 regulates virus release. These mechanisms and structures will be correlated with those of other enveloped and non-enveloped viruses, including the ones cited above. PUBLIC HEALTH RELEVANCE: Viruses in the Reoviridae family are non-enveloped, dsRNA viruses that infect a wide range of hosts, including human, other animals, plants, fungi, and bacteria; within this family, reoviruses and rotaviruses infect humans, and BTV kills livestock, with devastating economic and social consequences. The proposed structural and functional studies focus on BTV to provide structural answers to fundamental questions of cell entry and transport of these and other non-enveloped dsRNA viruses. The results will provide profound insight into the structural basis of non-enveloped virus infection and reveal targets for rational development of anti-viral drugs and vaccines effective against these human and animal viruses.